Membrane tension is thought to be a long-range integrator of cell physiology, where it has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.
In this study, we used super-resolution Structured Illumination Microscopy (SIM) and Live Cell Confocal Microscopy to track the fate of RNA loaded in exosomes in MDA-MB-231 cells, a model for Triple Negative Breast Cancer. We employed a stable 3WJ RNA motif with a 3' end cholesterol modification for exosome decoration. Gel electrophoresis confirmed a 1:1000 binding ratio of exosomes to RNA-Cholesterol, and Zetasizer data showed that this decoration increased the exosomes' negative charge. Unlike prior studies, which focused on stained exosome membranes in lysosomes, we directly visualized the RNA payloads in both decorated and non-decorated exosomes. Using SIM, we confirmed the localization of the RNA within the exosome membrane and the RNA-Cholesterol on the exosome membrane. Within the first 24 hours, decorated exosomes aggregated less and primarily adhered to the cell membrane, an interaction observed at much higher rates in naked exosomes. By 48 hours, both types of exosomes showed significant lysosomal co-localization, with the rate accelerated in decorated exosomes. At 72 hours, the RNA payload was near or within the nucleus and diffused at low intensity throughout the cytosol, with higher fluorescent intensity observed in decorated exosomes. Lysosomal colocalization remained unchanged between 48-72 hours. Western blot analysis confirmed effective Survivin gene silencing at 72 hours, suggesting that lysosomal localization to some extent impedes exosomal RNAi delivery to cells. We propose that the RNA payload enters the cell through multiple pathways. Direct fusion between the exosome and cell membrane occurs at low frequencies, typically undetected in fluorescent microscopy until later stages where the net aggerate in the cytosol is detectable, but contributes to the majority of functional RNA observed in the cytosol and around the nucleus. RNA-Cholesterol decoration enhances the rate of fusion by de-aggregating the exosomes, allowing them to interact at a higher net quantity than naked exosomes, whereas the increased negative charge reduces affinity between the exosome and the cell membrane. Our work provides a framework by which exosomes payloads are directly observed interacting with cellular processes for functional delivery of RNAi and chemotherapeutics within cells using exosomes.
Probing DNA-protein complexes at single-molecule level has been interesting as it can provide insight into how biological machinery works. Many DNA nanodevices were created to probe biophysical properties of proteins and DNA-protein complexes, providing an accessible and reproducible way of monitoring and measurement. However, the sizes and sequences of these DNA nanodevices are defined by the phage genome as their scaffolds, limiting their size flexibility and making it hard to introduce DNA sequences of biological interest. Here, we used custom ssDNA production methods to build DNA origami nanodevices that use a block-tether-block design where the tether can carry DNA sequences of biological functionality like nucleosome positioning sequence. By attaching blocks to another rigid DNA origami platform, we are building a DNA origami nanodevice that can precisely control the position of dsDNA tether, or DNA-protein complex with protein bound to the tether. This is especially helpful to studies of inter-array interactions between reconstituted nucleosomes with its ability to control relative position of nucleosome arrays.
Last update: 10/10/2023, Ralf Bundschuh